Hepatitis C virus (HCV), a (+)-strand RNA virus, persistently infects ~200 million persons worldwide and, despite advances in antiviral therapy, remains the leading cause of hepatic cirrhosis and liver cancer in the U.S. In this application, we propose a continuation of a productive line of research aimed at understanding the molecular mechanism(s) underlying the unique dependence of HCV replication on the evolutionarily conserved, liver-specific host microRNA, miR-122-5p. Our published research shows that the binding of miR-122 to two conserved sites (S1 and S2) near the 5' end of the 9.7 kb (+)-sense RNA genome promotes HCV replication in two distinct ways: (1) by binding S1 and S2, miR-122 protects HCV RNA from 5' exonucleolytic decay mediated by XRN1, a cytoplasmic host 5' exoribonuclease, and (2) miR-122 directly stimulates HCV RNA synthesis by rebalancing the proportion of (+)-RNA molecules engaged in templating RNA synthesis vs. directing translation on ribosomes. Both functions require AGO2 which our data show is recruited to the genome by miR-122. We now show in new preliminary data that miR-122 also regulates circularization of the HCV genome through competition for binding with poly-(rC) binding protein 2 (PCBP2) and possibly other host RNA-binding proteins. We propose three specific aims to investigate the overarching hypothesis that a unique ribonucleoprotein complex formed by miR-122 and AGO2 at the 5' end of the HCV genome critically regulates its engagement in different stages of the viral lifecycle. In Aim 1, we will assess the role of miR-122 in regulating strand-specific HCV RNA synthesis and packaging of newly synthesized (+)-RNA. Aim 2 will study how viral replication is regulated by competition between miR-122 and host RNA-binding proteins, including PCBP2 and newly recognized candidate host factors identified through quantitative proteomics. Aim 3 will characterize the role of miR-122 in modulating circularization of the HCV (+)-strand genome mediated by protein-protein interactions, and how this relates to genome stability and viral RNA synthesis. While our efforts focus on HCV specifically, these studies have broad relevance to mechanisms of replication of other pathogenic (+)-strand RNA viruses.